Since the inception of this program proiect grant it has been the long-term goal of Project II to bring about a fuller understanding of the complex mechanisms that govern the tissue responses to Photodynamic Therapy (PDT). The Project Leader's laboratory has developed extensive expertise in the study of two major elements of the PDT tissue response: the effects of PDT on tumor oxygenation, and the closely connected effects on the host inflammatory/immune responses directed against the tumor. This renewal application draws on novel discoveries that have emerged from both of these areas of interest. These are (i) that exposure of tumors to PDT greatly affects the expression of a wide spectrum of molecules important for tumor growth; (ii) that PDT regimens can be devised that greatly differ in the type of tumor microenvironment they create, and thus can exert markedly different influences on the regulation of such molecules. Based on these discoveries we have formulated the following hypotheses: (1) The rate at which photodynamic damage occurs will influence the expression of stress-induced genes. (2) The status of tumor oxygenation during and after PDT, largely determined by the parameters of a given PDT regimen, will affect the redoxdependent regulation of molecules important for tumor growth and/or anti-tumor host responses; (3) The mechanism of cell death within the tumor, influenced by a given photosensitizer as well as the microenvironmental conditions created by the PDT regimen will determine the "danger signals" generated and thus will influence tumor cell and anti-tumor host responses. To test these hypotheses we propose the following specific aims: Aim 1 To rationally devise PDT regimens in preclinical tumor models, based on our current knowledge of the different factors important for the PDT response (type of cell death, oxygenation) that are expected to create markedly different tumor microenvironments. Aim 2 To characterize the tumor milieus created by these PDT regimes, with special emphasis on signaling molecules that modulate the inflammatory/immune/angiogenic host response. Aim 3 To determine whether how host cells respond to these signals. Aim 4 To establish the molecular mechanisms by which PDT determines cytokine responsiveness of target cells. Our experilnental approach can be summarized as follows: (1) We will create different tumor microenvironments by, devising PDT regimes utilizing photosensitizers that induce different cell death pathways and by varying light fhience rates that affect tumor oxygenation, (2) We will use molecular techniques (microarray, RT-PCIL. Western blotting. ELISA) to characterize changes in protein expression induced by these PDT regimes; (3) We will assess the response of tumor and host to these changes by employing flow cytometry, immunohistochemistry, adhesion assays to assess host cell recruitment and apoptosis; (4) We will employ immunoblotting for detection of proteins, protein modifications and status of phosphorylation, immunoprecipitation, RT-PCR and Northern blot analyses for mRNA and flow cytometie assessment of cell cycle stage, apoptosis and marker protein expression to determine PDT effects on cytokine signaling. Our long-standing collaboration and multi-disciplinary approach aims to gain a comprehensive view of the mechanisms determining PDT tumor response, thereby possibly developing new treatment paradigms and optimizing treatment design. The subprojects are tightly, linked as they are complementary and necessary extensions of each other. Projects II will provide important input into clinical study design proposed in Project IV. The project is supported by three scientific Cores.